We are analyzing gene expression patterns in leukemia cells from patients with chronic lymphocytic leukemia (CLL) that we obtain from peripheral blood, bone marrow and lymph nodes. Our hypothesis is that the leukemic cells receive essential proliferation and survival signals in the bone marrow and/or lymph node. We have analyzed 8 matched pairs of bone marrow and blood derived CLL cells. Consistent with our hypothesis we found that CLL cells from the bone marrow showed a higher expression of genes associated with proliferation as well as a couple hundred genes that may relate to the specific signals induced in the leukemic cells by specific signals provided by the bone marrow microenvironment. We are extending this study now to lymph node samples and are following up on candidate molecules that could play a role in stimulating CLL cell survival. In addition, we are establishing models in vitro where we can show that the survival of CLL cells is extended by co-culture with strom cells. From these studies we hope to identify which signaling pathways are essential for leukemic cell survival and which therefore could be good targets of new therapies.[unreadable] [unreadable] In a second part of the project we obtain CLL cells from patients who undergo therapy with two of the most active drugs in CLL therapy; rituximab and fludarabine. This therapy is given over a 6 day period and repeated every 4 weeks for 6 cycles. Patients donate blood every day during the first 6 treatment days and we analyze the changes in gene expression in these cells due to the therapy. Rituximab, a monoclonal anti-CD20 antibody, is used to treat Chronic Lymphocytic Leukemia (CLL) in combination with fludarabine. Rituximab is thought to deplete B-cells through antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and possibly signaling for apoptosis. Whether or not signaling by rituximab contributes to its clinical efficacy and can sensitize the malignant cells to chemotherapy is controversial. To investigate if rituximab can induce a specific gene expression signature, we used gene expression profiling of B-cells from CLL patients receiving their first rituximab infusion. During the infusion, patients experienced a cytokine release syndrome (fever, chills, and hypotension) that led to interruption and symptomatic treatment in most; however, all patients were able to finish the treatment. We so far have analyzed CD19+ selected CLL cells from eight patients obtained pre and 6 and 24 hours after the start of rituximab. We identified a distinct set of genes important in immune regulation and inflammation that were upregulated in response to rituximab. The majority of these genes were at least 2-fold up-regulated at 6 hours, but most returned to pre-treatment levels by 24 hours. Thus, rituximab induced a transient gene expression signature that correlated with the cytokine release syndrome during the infusion. Ongoing studies aim to better characterize rituximab signaling in CLL and to determine whether this can contribute to apoptosis or sensitize the leukemic cells to chemotherapy.These results will improve our understanding on how these drugs kill the leukemic cells and may help to further improve therapeutic drug combinations.